PROJECT SUMMARY/ABSTRACT Misregulation of cell fate and identity is at the crux of human developmental diseases, including neurological disorders and cancer. The transition from one cell identity to another along a developmental trajectory is a key control point, yet it is largely unclear how these transitions and cell identities are regulated. To investigate fundamental principles that direct stem cell fate and identity, this proposal will examine the multipotent stem cell stomatal lineage in the model system Arabidopsis thaliana. Stomata, whose name derives from ?mouth? in Greek, are specialized organs on the surface of the leaf that are comprised of two cells, called guard cells. Guard cells, and thus stomata, form a pore that opens and closes to enable the exchange of gases with the atmosphere. Stomata arise from three discrete cell lineage states (i-iii) that are sequentially driven by three conserved bHLH transcriptional regulators: SPCH, MUTE, and FAMA. Initially, a series of asymmetric cells divisions that mediate stem cell self-renewal (i) are orchestrated by SPCH, which is followed by a subsequent cell differentiation event (ii) controlled by MUTE. A final symmetric cell division and maturation event (iii) is driven by FAMA. Since plant cells are locked in place by their cell walls and the stomatal lineage is found in the epidermal plane, neighboring cells tell the story of their past divisions, providing a nearly unrivaled opportunity to examine mechanisms that underlie stem cell fate and identify decisions. Furthermore, the number of stomata interspersed among interdigitated epidermal cells varies in response to environmental cues. This feature enables us to examine the range of adaptable regulatory logic in a multipotent stem cell lineage through simple manipulations of environmental cues. Thus, the stomatal lineage provides key advantages ? the ability to readily analyze and visualize dividing and differentiating cells in an intact, developmentally adaptable tissue. This proposal thus integrates single-cell systems biology with whole-tissue developmental biology and molecular genetics approaches to test our central hypothesis that the stomatal lineage consists of lineally related heterogeneous and adaptable cell states. Aim 1 will analyze the range of heterogeneity in the stomatal cell lineage, which should extend our understanding of conserved pathways in stomatal development. Aim 2 will determine molecular mechanisms that confer lineage adaptability (thereby rendering it ?flexible?), and it should reveal insight into molecular mechanisms that promote flexible cell fate decisions. Together, the proposed experiments should illuminate generalizable strategies in cell fate and re-programming that may be used to promote human health and ameliorate developmental disorders.